Arteries were cleaned of perivascular adipose tissue, incubated with LXA4 (10 nmol/L; 15 min), snapped frozen in liquid nitrogen, and then homogenized in ice-cold tissue protein extraction reagent (T-PER) (Thermo Fisher Scientific, Waltham), with protease inhibitors [sodium orthovanadate, phenylmethanesulfonylfluoride (PMSF), protease inhibitor cocktail (Sigma)] and phosphatase inhibitors [sodium fluoride and sodium pyrophosphate] (Sigma). Protein was extracted and quantified equal amounts (20-40 μg) were separated on polyacrylamide gels using a standard SDS-PAGE western blot protocol.
The membranes were blocked with 5 % nonfat dry milk and incubated overnight at 4 °C with primary antibodies raised against anti-COX-1 and COX-2 (1:2000; Upstate), anti-p67phox (1:1000, Cell Signaling); phosphorylated myosin light chain Thr18/Ser19 (pMLC, 1:2000; Cell Signaling) and RhoA (1:2000; Cell Signaling).
The membranes were then washed and incubated with horseradish peroxidase-linked secondary antibodies. Immunocomplexes were detected with an enhanced chemiluminescence system on a protein simple imager. Phosphorylated protein expression was normalized to total protein expression; all other proteins were normalized to loading control, β-actin (Sigma-Aldrich). Expression is presented as arbitrary units (A.U.).
The membranes were blocked with 5 % nonfat dry milk and incubated overnight at 4 °C with primary antibodies raised against anti-COX-1 and COX-2 (1:2000; Upstate), anti-p67phox (1:1000, Cell Signaling); phosphorylated myosin light chain Thr18/Ser19 (pMLC, 1:2000; Cell Signaling) and RhoA (1:2000; Cell Signaling).
The membranes were then washed and incubated with horseradish peroxidase-linked secondary antibodies. Immunocomplexes were detected with an enhanced chemiluminescence system on a protein simple imager. Phosphorylated protein expression was normalized to total protein expression; all other proteins were normalized to loading control, β-actin (Sigma-Aldrich). Expression is presented as arbitrary units (A.U.).